Throughout this application various publications are referenced within parentheses. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains.
1. The Field of the Invention
This invention relates to the medical arts, particularly to the field of transgenics and gene therapy The invention is particularly directed to in vitro and in vivo methods for transfecting male germ cells and support cells (i.e., Leydig and Sertoli cells), which methods incorporate a method of depopulating a vertebrate testis of male germ cells.
2. Discussion of the Related Art
The field of transgenics was initially developed to understand the action of a single gene in the context of the whole animal and phenomena of gene activation, expression, and interaction. This technology has been used to produce models for various diseases in humans and other animals. Transgenic technology is amongst the most powerful tools available for the study of genetics, and the understanding of genetic mechanisms and function It is also used to study the relationship between genes and diseases About 5,000 diseases are caused by a single genetic defect. More commonly, other diseases are the result of complex interactions between one or more genes and environmental agents, such as viruses or carcinogens. The understanding of such interactions is of prime importance for the development of therapies, such as gene therapy and drug therapies, and also treatments such as organ transplantation Such treatments compensate for functional deficiencies and/or may eliminate undesirable functions expressed in an organism. Transgenesis has also been used for the improvement of livestock, and for the large scale production of biologically active pharmaceuticals.
Historically, transgenic animals have been produced almost exclusively by micro injection of the fertilized egg. The pronuclei of fertilized eggs are micro injected in vitro with foreign, i.e. xenogeneic or allogeneic DNA or hybrid DNA molecules. The micro injected fertilized eggs are then transferred to the genital tract of a pseudopregnant female. (E.g., P. J. A. Krimpenfort et al., Transgenic mice depleted in mature T-cells and methods for making transgenic mice, U.S. Pat. Nos. 5,175,384 and 5,434,340; P. J. A. Krimpenfort et al., Transgenic mice depleted in mature lymphocytic cell-type, U.S. Pat. No. 5,591,669).
The generation of transgenic animals by this technique is generally reproducible, and for this reason little has been done to improve on it. This technique, however, requires large numbers of fertilized eggs. This is partly because there is a high rate of egg loss due to lysis during micro injection. Moreover manipulated embryos are less likely to implant and survive in utero. These factors contribute to the technique""s extremely low efficiency. For example, 300-500 fertilized eggs may need to be micro injected to produce perhaps three transgenic animals. Partly because of the need to micro inject large numbers of embryos, transgenic technology has largely been exploited in mice because of their high fecundity. Whilst small animals such as mice have proved to be suitable models for certain diseases, their value in this respect is limited. Larger animals would be much more suitable to study the effects and treatment of most human diseases because of their greater similarity to humans in many aspects, and also the size of their organs. Now that transgenic animals with the potential for human xenotransplantation are being developed, larger animals, of a size comparable to man will be required. Transgenic technology will allow that such donor animals will be immunocompatible with the human recipient. Historical transgenic techniques, however, require that there be an ample supply of fertilized female germ cells or eggs. Most large mammals, such as primates, cows, horses and pigs produce only 10-20 or less eggs per animal per cycle even after hormonal stimulation. Consequently, generating large animals with these techniques is prohibitively expensive.
This invention relies on the fact that vast numbers of male germ cells are more readily available. Most male mammals generally produce at least 108 spermatozoa (male germ cells) in each ejaculate. This is in contrast to only 10-20 eggs in a mouse even after treatment with superovulatory drugs. A similar situation is true for ovulation in nearly all larger animals. For this reason alone, male germ cells will be a better target for introducing foreign DNA into the germ line, leading to the generation of transgenic animals with increased efficiency and after simple, natural mating.
Spermatogenesis is the process by which a diploid spermatogonial stem cell provides daughter cells which undergo dramatic and distinct morphological changes to become self-propelling haploid cells (male gametes) capable, when fully mature, of fertilizing an ovum.
Primordial germ cells are first seen in the endodermal yolk sac epithelium at E8 and are thought to arise from the embryonic ectoderm (A. McLaren and Buehr, Cell Diff. Dev. 31:185 [1992]; Y. Matsui el al., Nature 353:750 [1991]). They migrate from the yolk sac epithelium through the hindgut endoderm to the genital ridges and proliferate through mitotic division to populate the testis.
At sexual maturity the spermatogonium goes through 5 or 6 mitotic divisions before it enters meiosis. The primitive spermatogonial stem cells (Ao/As) proliferate and form a population of intermediate spermatogonia types Apr, Aal, A1-4 after which they differentiate into type B spermatogonia. The type B spermatogonia differentiate to form primary spermatocytes which enter a prolonged meiotic prophase during which homologous chromosomes pair and recombine. The states of meiosis that are morphologically distinguishable are; preleptotene, leptotene, zygotene, pachytene, secondary spermatocytes and the haploid spermatids. Spermatids undergo great morphological changes during spermatogenesis, such as reshaping the nucleus, formation of the acrosome and assembly of the tail (A. R. Bellve et al, Recovery, capacitation, acrosome reaction, and fractionation of sperm, Methods Enzymol. 225:113-36 [1993]). The spermatocytes and spermatids establish vital contacts with the Sertoli cells through unique hemi-junctional attachments with the Sertoli cell membrane. The final changes in the maturing spermatozoan take place in the genital tract of the female prior to fertilization.
Initially, attempts were made to produce transgenic animals by adding DNA to spermatozoa which were then used to fertilize mouse eggs in vitro. The fertilized eggs were then transferred to pseudopregnant foster females, and of the pups born, 30% were reported to be transgenic and express the transgene. Despite repeated efforts by others, however, this experiment could not be reproduced and no transgenic pups were obtained. Indeed, there remains little doubt that the transgenic animals reputed to have been obtained by this method were not transgenic at all and the DNA incorporation reported was mere experimental artifact. Data collected from laboratories around the world engaged in testing this method showed that no transgenics were obtained from a total of 890 pups generated.
In summary, it is currently possible to produce live transgenic progeny but the available methods are costly and extremely inefficient. Spermatogenic transfection in accordance with this invention, either in vitro or in vivo, provides a simple, less costly and less invasive method of producing transgenic animals and one that is potentially highly effective in transferring allogeneic as well as xenogeneic genes into the animal""s germ cells.
To facilitate in vitro transfection of male germ cells and implantation into a testis of a recipient male vertebrate it is advantageous first to depopulate the testis of the recipient vertebrate of untransfected male germ cells before transferring transfected male germ cells into it.
Depopulation oftestis has commonly been done by exposing the whole vertebrate to gamma irradiation (X-ray), or localizing irradiation to the testis. (E.g., G. Pinon-Lataillade et al., Endocrinological and histological changes induced by continuous low dose gamma-irradiation of rat testis, Acta Endocrinol. (Copenh) 109(4):558-62 [1985]; G. Pinon-Lataillade and J. Maas, Continuous gamma-irradiation of rats: dose-rate effect on loss and recovery of spermatogenesis, Strahlentherapie 161(7):421-26 [1985]; C. R. Hopkinson et al., The effect of local testicular irradiation on testicular histology and plasma hormone levels in the male rat, Acta Endocrinol. (Copenh) 87(2):413-23 [1978]; G. Pinon-Lataillade et al, Influence of germ cells upon Sertoli cells during continuous low-dose rate gamma-irradiation of adult rats, Mol. Cell Endocrinol. 58(1):51-63 [1988]; P. Kamtchouing et al, Effect of continuous low-dose rate gamma-irradiation on rat Sertoli cell function, Reprod. Nutr. Dev. 28(4B):1009-17 [1988]; C. Pineau et al, Assessment of testicular function after acute and chronic irradiation: further evidence for influence of late spermatids on Sertoli cell function in the adult rat, Endocrinol. 124(6):2720-28 [1989]; M. Kangasniemi et al., Cellular regulation of basal and FSH-stimulated cyclic AMP production in irradiated rat testes, Anat. Rec. 227(1):32-36 [1990]; G. Pinon-Lataillade et al, Effect of an acute exposure of rat testes to gamma rays on germ cells and on Sertoli and Leydig cell functions, Reprod. Nutr. Dev. 31(6):617-29 [1991]).
The mechanism of gamma radiation-induced spermatogonial degeneration is thought to be related to the process of apoptosis. (M. Hasegawa et al., Resistance of differentiating spermatogonia to radiation-induced apoptosis and loss in p53-deficient mice, Radiat. Res.149:263-70 [1998]).
Another method of depopulating a vertebrate testis is by administering a composition containing an alkylating agent, such as busulfan (Myleran). (E.g., F. X. Jiang, Behaviour of spermatogonia following recovery from busulfan treatment in the rat, Anat. Embryol. 198(1):53-61 [1998]; L. D. Russell and R. L. Brinster, Ultrastructural observations of spermatogenesi following transplantation of rat testis cells into mouse seminiferous tubules, J. Androl. 17(6):615-27 [1996]; N. Boujrad et al, Evolution of somatic and germ cell populations after busulfan treatment in utero or neonatal cryptochidism in the rat, Andrologia 27(4):223-28 [1995]; R. E. Linder et al, Endpoint of spermatotoxicity in the rat after short duration exposures to fourteen reproductive toxicants, Reprod. Toxicol. 6(6):491-505 [1992]; F. Kasuga and M. Takahashi, The endocrine function of rat gonads with reduced number of germ cells following biusulfan treatment, Endocrinol. Jpn 33(1):105-15 [1986]).
Cytotoxic alkylating agents, such as busulfan, chlorambucil, cyclophosphamide, melphalan, or ethyl ethanesulfonic acid, are frequently used to kill malignant cells in cancer chemotherapy. (E.g., Andersson et al, Parenteral busutfan for treatment of malignant disease, U.S. Pat. Nos. 5,559,148 and 5,430,057; Stratford et al., Stimulation of stem cell growth by the bryostatins, U.S. Pat. No. 5,358,711; Luck et al., Treatment employing vasoconstrictive substances in combination with cytotoxic agents for introduction into cellular lesion, U.S. Pat. No. 4,978,332). Treatment of mice with busulfan (13 mg-40 mg/kg body wt.), was reported to deplete male germs cells in the testis; both stems cells and differentiating spermatogonia were killed; doses over 30 mg/kg body weight resulted in azoospermia for up to 56 days after treatment. (L. R. Bucci and M. L. Meistrich, Effects of busulfan on murine spermatogenesis: cytotoxicity, sterility, sperm abnormalities and dominant lethal mutations, Radiation Research 176:259-68 [1987]).
The present invention addresses the need for spermatogenic transfection, either in vitro or in vivo, that is highly effective in transferring allogeneic as well as xenogeneic genes into the animal""s germ cells and in producing transgenic vertebrate animals. The present technology addresses the requirements of germ line and stem cell line gene therapies in humans and other vertebrate species, including the need for a superior method of depopulating a testis of untransfected male germ cells. The present technology is of great value in producing transgenic animals in large species as well as for repairing genetic defects that lead to male infertility. Male germ cells that have stably integrated the DNA are selectable.
These and other benefits and features of the present invention are described herein.
The present invention relates to the in vivo and ex vivo (in vitro) transfection of eukaryotic animal germ cells with a desired genetic material. Briefly, the in vivo method involves injection of genetic material together with a suitable vector directly into the testicle of the animal. In this method, all or some of the male germ cells within the testicle are transfected in situ, under effective conditions. The ex vivo method involves extracting germ cells from the gonad of a suitable donor or from the animal""s own gonad, using a novel isolation method, transfecting or otherwise genetically altering them in vitro, and then returning them to the testis under suitable conditions where they will spontaneously repopulate it. The ex vivo method has the advantage that the transfected germ cells may be screened by various means before being returned to the testis to ensure that the transgene is incorporated into the genome in a stable state. Moreover, after screening and cell sorting only enriched populations of germ cells may be returned. This approach provides a greater chance of transgenic progeny after mating.
This invention also relates to a novel method for the isolation of spermatogonia, comprising obtaining spermatogonia from a mixed population of testicular cells by extruding the cells from the seminiferous tubules and gentle enzymatic disaggregation. The spermatogonia or stem cells which are to be genetically modified, may be isolated from a mixed cell population by a novel method including the utilization of a promoter sequence, which is only active in cycling spermatogonial stem cell populations, for example, B-Myb or a spermotogonia specific promoter, such as the c-kit promoter region, c-raf-1 promoter, ATM (ataxia-telangiectasia) promoter, RBM (ribosome binding motif) promoter, DAZ (deleted in azoospermia) promoter, XRCC-1 promoter, HSP 90 (heat shock gene) promoter, cyclin A1 promoter, or FRMI (from fragile X site) promoter, optionally linked to a reporter construct, for example, a construct encoding Green Fluorescent Protein ([GFP] or enhanced GFP [EGFP]), Yellow Fluorescent Protein, Blue Fluorescent Protein, a phycobiliprotein, such as phycoerythrin or phycocyanin, or any other protein which fluoresces under suitable wave-lengths of light, or encoding a light-emitting protein. These unique promoter sequences drive the expression of the reporter construct only in the cycling spermatogonia. The spermatogonia, thus, are the only cells in the mixed population which will express the reporter construct and they, thus, may be isolated on this basis. Transgenic cells expressing a fluorescent or luminescent reporter construct can be sorted with the aid of, for example, a flow activated cell sorter (FACS) set at the appropriate wavelength or they may be selected by chemical methods.
The invention also relates to an effective method of substantially depopulating a vertebrate testis of male germ cells. The method involves administering a combination of a dose of an alkylating agent, such as busulfan, and a dose of gamma radiation to a vertebrate animal in an amount sufficient to substantially depopulate the vertebrate testis, to prepare it for implantation of male germ cells from a donor animal, for example. This combined treatment with an alkylating agent and gamma irradiation yields histologically superior results in eliminating the population of native untransfected or genetically unaltered male germ cells, compared to either an alkylating agent or gamma irradiation alone. Therefore, the present method of depopulating a vertebrate testis maximizes the production of transgenic animals using the present in vitro method of incorporating a polynucleotide encoding a desired trait or product into a maturing male germ cell.
This invention also relates to the repopulation of a testis with germ cells that have been isolated from a fresh or frozen testicular biopsy. These germ cells may or may not be genetically manipulated prior to implantation into a recipient testis.
For transfection (i.e., gene delivery), the method of the invention comprises administering to the animal, or to germ cells in vitro, a composition comprising amounts of nucleic acid comprising polynucleotides encoding a desired trait or product. In addition, the composition comprises, for example, a relevant controlling promoter region made up of nucleotide sequences. This is combined with, for example, a gene delivery system comprising a cell transfection promotion agent such as retro viral vectors, adenoviral and adenoviral related vectors, or liposomal reagents or other agents used for gene therapy. These introduced under conditions effective to deliver the nucleic acid segments to the animal""s germ cells optionally with the polynucleotide inserted into the genome of the germ cells. Following incorporation of the DNA, the treated animal is either allowed to breed naturally, or reproduced with the aid of assisted reproductive technologies, and the progeny selected for the desired trait.
This technology is applicable to the production of transgenic animals for use as animal models, and to the modification of the genome of an animal, including a human, by addition, modification, or subtraction of genetic material, often resulting in phenotypic changes. The present methods are also applicable to altering the carrier status of an animal, including a human, where that individual is carrying a gene for a recessive or dominant gene disorder, or where the individual is prone to pass a multigenic disorder to his offspring.
A preparation suitable for use with the present methods comprises a polynucleotide segment encoding a desired trait and a transfection promotion agent, and optionally an uptake promotion agent which is sometime equipped with agents protective against DNA breakdown. The different components of the transfection composition are also provided in the form of a kit, with the components described above in measured form in two or more separate containers. The kit generally contains the different components in separate containers. Other components may also be provided in the kit as well as a carrier.